Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Strategy to detect pre-existing immunity to AAV gene therapy.

Gene therapy may offer a new treatment option, particularly for patients with severe hemophilia, based on recent research. However, individuals with pre-existing immunity to adeno-associated viruses (AAVs) may be less likely to benefit from AAV vector-based therapies. To study pre-existing AAV5 immunity in humans, we validated two complementary, sensitive, and scalable in vitro assays to detect AAV5 total antibodies and transduction inhibition (TI). Using these two assays, we found that 53% of samples from 100 healthy male individuals were negative in both assays, 18% were positive in both assays, 5% were positive for total antibodies but negative for TI and, of interest, 24% were negative for total antibodies but positive for TI activity, suggesting the presence of non-antibody-based neutralizing factors in human plasma. Similar findings were obtained with 24 samples from individuals with hemophilia A. On the basis of these results, we describe the development of a dual-assay strategy to identify individuals without total AAV5 antibodies or neutralizing factors who may be more likely to respond to AAV5-directed gene therapy. These assays offer a universal, transferrable platform across laboratories to assess the global prevalence of AAV5 antibodies and neutralizing factors in large patient populations to help inform clinical development strategies.

Physical presence during gamma stereotactic radiosurgery.

During an invited visit to the University of Pittsburgh Medical Center (UPMC) gamma knife facility, officials from the Nuclear Regulatory Commission (NRC) observed what they considered as an apparent violation of the physical presence requirements specified in 10 CFR 35.615(f)(3). This event initiated an inspection and two different but related investigations by the NRC Office of Investigations (OI). Based on the NRC inspection and investigations, the NRC identified three apparent violations that were under consideration for escalated enforcement. The University of Pittsburgh (licensee) requested an Alternative Dispute Resolution (ADR) session with the NRC to resolve issues related to whether a violation occurred, the appropriate enforcement action, and the appropriate corrective action. As a result of the ADR mediation session, the licensee and NRC agreed to final disposition of this matter by way of a single violation of the regulatory requirement in 10 CFR 35.24(b), whereby the licensee's Radiation Safety Officer failed to ensure that the physical presence requirements of 10 CFR 35.615(f)(3) were consistently met and failed to ensure that written directives were consistently signed by the Authorized User in accordance with 10 CFR 35.32. In addition to corrective actions the licensee had already taken to prevent recurrence, it also agreed to inform other licensees in the industry of this event, so that they may learn from this incident and take appropriate actions to assure that these types of violations do not occur at their institutions.

Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation.

In the present study, 4 patients with cystic fibrosis undergoing lung transplantation (from a total of 137) who developed fulminant pseudomembranous colitis are described. Initial presentation was variable and the mortality rate was 50% despite urgent colectomy. In one case the presenting abdominal distension was thought to be due to meconium ileus equivalent. It is concluded that Clostridium difficile colitis may be a difficult diagnosis in patients with cystic fibrosis and follows a fulminant course after lung transplantation.

Prevalencia y características de la hiperglicemia incidental en niños / Prevalence and characteristics of incidental hyperglycemia in children

Se describe la hiperglicemia incidental (HI) como una elevación transitoria de los niveles plasmáticos de la glucosa producida por una enfermedad intercurrente seria o sin causa aparente. Los niños con HI tienen un mayor riesgo de diabetes tipo 1, especialmente los sin enfermedad intercurrente. Nos interesó conocer la prevalencia de HI en niños sanos y sus características clínicas. En 585 niños de 2 a 18 años de edad que consultaban por control de salud o por cuadro infeccioso menor o antes de cirugía menor electiva, previo consentimiento del apoderado, se les efectuó una glicemia en sangre capilar con una máquina Optium (Abbott). Se consideró elevada una glicemia en ayunas sobre 110 mg/dL. El peso se midió con una balanza de precisión, la talla con un cartabón, los pliegues cutáneos con un Caliper de Lange, el % de grasa corporal se calculó por la fórmula de Durnin. Se encontró que un 6.9% de los niños presentó una glicemia sobre 110 mg/dL (8.8% de varones y 4.7% de mujeres). El promedio de peso y talla de nacimiento y edad gestacional fueron normales. El % de adecuación del IMC fue de 123.4%. Los 39 niños con HI comparados con los normales no tuvieron diferencias en el peso y talla de nacimiento, en las semanas de gestación, en el % de adecuación del IMC (114 vs 112%), en el % de grasa corporal. Sus madres no tuvieron mas antecedentes de diabetes previa o gestacional. Tuvieron mayor frecuencia de acantosis nigricans (9.5% vs 0.6% p< 0.001) y de estrías cutáneas (18% vs 3.0% p <0.001). Se concluye que existe un alto porcentaje de HI en una población de niños sanos, los que no presentaron mas antecedentes de riesgo de diabetes tipo 1 que los controles, pero presentaron mas acantosis y estrías de distensión que indicarían junto a los altos niveles de exceso de peso, una mayor posibilidad de hacer resistencia a la insulina y diabetes tipo 2, lo que sería de interés investigar.

Incidental hyperglycemia (IH) is a transient elevation of plasma glucose produced by a severe illness or without a cause. Children with IH have an increased risk of type 1 diabetes (T1D), especially those without concurrent illnesses. We were interested in knowing the prevalence of IH in a population of healthy children and their clinical characteristics. A glycemia of capillary blood with an Optium machine (Abbott) was done in 585 children of both sexes, aged 2 to 18 years, going for a health control, a light infectious disease or for clinical test before minor surgery after obtaining a written consent from their parents. A fasting blood glycemia over 110 mg/dl was considered elevated. Body weight was measured with a precision scale, height with a podometer, skinfolds with a Lange Caliper and the percentage of body fat calculated by the Durnin formula. It was found that 6.9% of children presented IH (8.85% of boys and 4.7% of girls). Average birth weight and height and gestational age were normal. BMI adequacy was 123 %. Comparing the 39 children who presented IH with those who id not, they had no differences in birth weight and length, weeks of gestational age, in BMI adequacy (114% vs 112%) and in the percentage of body fat. Their mothers did not have a past history of previous diabetes or diabetes during pregnancy. IH children had increased frequency of acantosis nigricans (9.5% vs 0.6%) and dermic striae (18% vs 3.0%) when compared to control. We conclude that a high percentage of healthy children had IH without increase risk of T1D. However, increased prevalence of acantosis and striae and the increased BMI underline the possibility of developing insulin resistance and T2D which should be further investigated.

Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation.

Pseudomembranous colitis is an uncommon complication in patients with cystic fibrosis, despite the use of multiple high-dose antibiotic regimens and the frequency of hospital admissions. Four patients from a total of 137 patients with cystic fibrosis undergoing lung transplantation are described who developed fulminant pseudomembranous colitis. Initial presentation was variable and the mortality rate was 50% despite urgent colectomy. In one case the presenting abdominal distension was thought to be due to meconium ileus equivalent. It is concluded that Clostridium difficile colitis may be a difficult diagnosis in patients with cystic fibrosis and follows a fulminant course after lung transplantation.

Plasma hormone levels and central c-Fos expression in ferrets after systemic administration of cholecystokinin.

Posterior pituitary hormone secretion and central neural expression of the immediate-early gene product c-Fos was examined in adult ferrets after intravenous administration of CCK octapeptide. Pharmacological doses of CCK (1, 5, 10, or 50 microg/kg) did not induce emesis, but elicited behavioral signs of nausea and dose-related increases in plasma vasopressin (AVP) levels without significant increases in plasma oxytocin (OT) levels. CCK activated neuronal c-Fos expression in several brain stem viscerosensory regions, including a dose-related activation of neurons in the dorsal vagal complex (DVC). Activated brain stem neurons included catecholaminergic and glucagon-like peptide-1-positive cells in the DVC and ventrolateral medulla. In the forebrain, activated neurons were prevalent in the paraventricular and supraoptic nuclei of the hypothalamus and also were observed in the central nucleus of the amygdala and bed nucleus of the stria terminalis. Activated hypothalamic neurons included cells that were immunoreactive for AVP, OT, and corticotropin-releasing factor. Comparable patterns of brain stem and forebrain c-Fos activation were observed in ferrets after intraperitoneal injection of lithium chloride (LiCl; 86 mg/kg), a classic emetic agent. However, LiCl activated more neurons in the area postrema and fewer neurons in the nucleus of the solitary tract compared with CCK. Together with results from previous studies in rodents, our findings support the view that nauseogenic treatments activate similar central neural circuits in emetic and nonemetic species, despite differences in treatment-induced emesis and pituitary hormone secretion.

A study of titanium nitride diffusion barriers between aluminum and silicon by X-ray absorption spectroscopy: the Si, Ti and N results.

We report a multi-elment, multi-edge and multi-detection mode X-ray photoabsorption study of a series of Al/TiN(x)/Si(100) thin films as a function of the TiN(x) film thickness (100A-500A) and of the annealing temperature (400 degrees C-600 degrees C). The Si K- and L-edge results show that Si does not diffuse to the surface for all the films. The high resolution Ti L-edge and N K-edge spectra show that the TiN(x) layer undergoes a dramatic chemical reaction with the gradual increase in the annealing temperature. This chemical reaction stabilizes at 560 degrees C at which the TiN(x) film is known to fail to act as an effective diffusion barrier between Al and Si.

Angioleiomyoma: clinical presentation and surgical management.

Angioleiomyomas are relatively rare, benign, vascular soft tissue tumors with a predilection for the lower extremity. They may occur in either the cutaneous or subcutaneous tissue and are usually well encapsulated. Erosion of adjacent bone due to secondary compression phenomenon has rarely been reported in the literature. This study describes three cases.

Concordance of risk factors in female spouses of male patients with coronary heart disease.

PURPOSE: Shared environment may put marital partners at increased risk for coronary heart disease (CHD). In this study, the authors examined the degree of concordance of risk factors between men with CHD (n = 177) and their spouses, and described the risk profiles of both patients and spouses. Risk factors examined were smoking, hypertension, obesity, cholesterol level, diet, and exercise. METHODS: Data were collected 2 months after the cardiac event using the Behavioral Risk Factor Surveillance System tool. Concordance between patient and spouse pairs was evaluated using Pearson correlation coefficients (r) for continuous data and phi coefficients (phi) for nominal data. RESULTS: Significant concordance was found between patient and spouse pairs for body mass index, history of smoking, current smoking status, frequency of exercise, miles per exercise session, and the amount of fat and fiber in the diet. There was no significant spousal concordance in relation to the diagnosis of hypertension, systolic and diastolic blood pressure (BP), cholesterol level, history of high cholesterol, current exercise program, duration of exercise, and amount of salt in the diet. Thus, although the physiological indicators of risk (e.g., BP) were not significantly related among marital partners, behavioral indicators of risk (e.g., smoking) were significantly related. CONCLUSIONS: The findings suggested that shared lifestyles of marital partners may result in greater risk of CHD for female partners of men with CHD. Furthermore, lifestyle interventions that specifically target the marital partners as a unit may be more efficacious than individual patient education strategies.

Quality of life comparisons after coronary angioplasty and coronary artery bypass graft surgery.

OBJECTIVE: To examine the differences in realization of expected benefits, complications, and quality of life (QOL) 3 months after percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft (CABG) surgery. DESIGN: Nonexperimental, prospective, and comparative. Before discharge, participants listed benefits expected from the procedure, as well as comorbid health problems (Charlson Comorbidity Index) and complications. At 3 months, they quantified their realization of expected benefits, reported postdischarge complications, and completed Ferrans and Powers' Quality of Life Index-Cardiac Version III. SAMPLE: 36 patients who had PTCA; 38 patients who had CABG. RESULTS: There were no differences between groups in realization of expected benefits or QOL. Patients who had CABG reported a greater number of complications after discharge, and a greater proportion of patients who had PTCA reported angina. Patients who had PTCA and then recurrent angina had significantly lower health QOL and psychologic and spiritual QOL. CONCLUSIONS: Patients who undergo CABG need guidance regarding what complications to expect, and patients who undergo PTCA need to know that recurrent angina is possible and how to manage it.

Improvements in clinical benefit with vinorelbine in the treatment of hormone-refractory prostate cancer: a phase II trial.

BACKGROUND: Clinical activity is difficult to assess by traditional response endpoints in patients with advanced prostate cancer. We used clinical benefit response to assess the activity of vinorelbine (Navelbine) in patients with hormone-refractory prostate cancer. PATIENTS AND METHODS: Forty-nine men with hormone-refractory prostate cancer received vinorelbine weekly for eight weeks followed by every-other-week dosing. Clinical benefit response was defined by improvement in 1 of the following categories for at least 12 weeks and stable response or better in the other 2: pain index (analgesic consumption and pain intensity), Karnofsky performance status, and tumor status. RESULTS: Of 37 evaluable patients, 14 (39%) achieved clinical benefit for a median duration of 6 months (range 3-24 months). Toxicities consisted primarily of brief neutropenia and mild nausea. CONCLUSION: These findings indicate that vinorelbine is well tolerated in men with hormone-refractory prostate cancer and produces durable clinical benefit as defined by improvement in pain index and performance status.

Autonomic reaction to vestibular damage.

The vestibular system provides inputs to many neurons in the brain stem that participate in autonomic control. This multiplicity of vestibular-autonomic connections plays a variety of roles. Whereas it has been known for decades that unilateral vestibular lesions can result in motion sickness, recent data suggest that the vestibular system participates in making adjustments in blood pressure and respiration that are necessary to maintain homeostasis during movement and changes in posture. Animals with bilateral vestibular lesions are more susceptible to posturally related hypotension than vestibularly intact animals, and it is also possible that orthostatic hypotension after space flight is caused in part by microgravity-related changes in otolith function. Patients with vestibular lesions could also be more vulnerable to respiratory disturbances related to posture, such as obstructive apnea. Vestibular dysfunction has additionally been linked with anxiety disorders, such as agoraphobia, which may result from alteration of vestibular inputs to brain stem monoaminergic neurons (which are known to process these signals). Even sleep disturbances might be connected with vestibular disorders because neurons in the pontine reticular formation that are critical in switching between sleep states may be influenced by labyrinthine inputs. Thus it is likely that vestibular damage will result in a number of parallel disturbances in autonomic function.

Comparison of psychologic stress responses in patients and spouses ten weeks after a cardiac illness event.

A stress scale that is brief and easy to administer would be clinically useful to assess patients' and spouses' psychological stress responses to a cardiac illness situation and provide objective data for health care recommendations. The purpose of this study was to: a) develop and test an instrument to evaluate the stress or difficulties associated with the recovery process after a cardiac event; and b) assess the similarities and differences between patients' and spouses' perceptions of stress. Stress was conceptualized to be reflected by two broad dimensions, namely environmental stress and individual stress. A correlational survey design was used to examine patients' (n = 180) and spouses' (n = 168) stress responses 10 weeks after the cardiac event. The findings suggested that the tool demonstrated satisfactory reliability and validity in measuring the patients' but to a lesser extent the spouses' stress responses. The three most significant stresses reported by most patients were uncertainty about if and when the illness might recur, not being able to participate in work or retirement activities, and adopting a low cholesterol diet. For the majority of spouses, the three most significant stresses were uncertainty about the husband's illness, increased demands within the marital relationship, and assuming more family roles. For those individuals trying to quit smoking or who quit immediately prior to the cardiac event, smoking was the most stressful change for patients and the second most stressful for spouses. Educational programs for cardiac patients and spouses need to provide information related to these stresses to offset the potentially adverse impact of stress and promote adjustment.

Restriction enzyme-induced DNA double-strand breaks as a model system for cellular responses to DNA damage.

To learn more about cellular responses to DNA double-strand breakage, we used three methods to assay cellular damage after treatment with a restriction enzyme that causes DNA double-strand breaks by cleaving at specific recognition sites in the DNA. Chinese hamster ovary cells were treated with increasing doses of Pvu II and studied for double-strand breakage, chromosomal aberration yield, and cell survival. The yield of DNA double-strand breaks, as measured by pulsed-field gel electrophoresis, increased at concentrations up to 500 units and saturated thereafter. The maximum yield of metaphase cells showing aberrant chromosomes was reached at 100 units and stayed constant up to 1,000 units. Although exchange-type aberrations saturated at approximately 4.5 per cell at 100 units, deletion-type aberrations appeared to increase at concentrations up to 500 units. Cell survival, as measured by colony-forming ability after Pvu II treatment, saturated at 100 units. The observed dose-response data are probably due to the saturation of accessible Pvu II cleavage sites within the cell. These data indicate that restriction enzymes induce the same DNA-damaging effects as many of the agents used in cancer treatment. Because the primary DNA lesion induced by restriction enzymes is known, they provide a unique opportunity to understand cellular responses to DNA damage and repair.

New integrated gas turbine CHP (combined heat and power) and incinerator plant.

Despite the complex nature of the project, the clients brief of a 14 month design and installation period was achieved within the approved budget of 2.5 million pounds. Early performance figures indicate that the scheme is on target to achieve the original payback of under four years. Queen Elizabeth Hospital: installation of integrated combined heat and power plant. Client: Central Birmingham Health Authority. Consulting Engineers/Project Managers: Yates, Edge and Partners. Architects: Temple Cox and Nichols. Structural Engineers: Peel and Fowler. Quantity Surveyor: West Midlands Regional Health Authority.

Social support for women during chronic illness: the relationship among sources and types to adjustment.

The purpose of this study was to explore who in the network provided what type of support in relation to psychosocial adjustment for women experiencing chronic illness. The Norbeck Social Support Questionnaire was administered to 125 chronically ill women, along with measures of depression (CES-D), family illness demands (Demands of Illness Inventory), marital quality (Spanier Dyadic Adjustment Scale), and family functioning (FACES-II). Repeated-measures ANOVA was used to examine the average amount of support from four main sources: partner, family, friends, and others. Women perceived more support from the partner than from any other source. Family members provided more affective support than friends or others. Friends provided more affirmation than family or others. After the partner, women reported confiding about their illness more to health care providers, counselors, or religious personnel than family or friends. Pearson correlation coefficients were computed for the amount of support from each source and the measures of individual, dyadic, or family adjustment. In general, affect, affirmation, and reciprocity from both the partner and family were associated with less depression, higher marital quality, and better family functioning.

Functional role of cysteine-146 in Escherichia coli thymidylate synthase.

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.